The present disclosure relates generally to methods for ergothioneine biosynthesis. More particularly, the present disclosure relates to methods for microbial ergothioneine biosynthesis.
Ergothioneine (ET) is a histidine betaine derivative with a thiol group attached to the C2 atom of the imidazole ring. As a thione tautomer, ET is a very stable antioxidant with unique properties. Unlike glutathione and ascorbate, ET can scavenge oxidizing species that are not free radicals. ET is a natural compound that is produced in Actinobacteria such as Mycobacterium smegmatis and filamentous fungi such as Neurospora crassa. Other species of bacteria, such as Bacillus subtilis, Escherichia coli, Proteus vulgaris and Streptococcus, as well as fungi belonging to the groups Ascomycetes and Deuteromycetes, cannot make ergothioneine. Animals and plants also cannot make ergothioneine and must obtain it from dietary sources or in the case of plants, from their environment.
Although the function of ET in microbial cells is not well understood, it is believed to be critical in human physiology. Humans absorb ET from dietary sources and ET accumulates in specific tissues and cells such as the liver, kidney, central nervous system, and red blood cells. It is evidenced that a specific cation transporter (OCTN1) has high affinity for ET in the human body, and both hyperactivity and deficiency of the transporter exert negative effects on human cells.
The biosynthesis of ET has been detected in certain mycobacteria fungi, however, the exact metabolic pathway is not completed or only partially confirmed. Seebeck reconstituted mycobacterial ergothioneine biosynthesis in vitro using E. coli to separately express a formylglycine-generating enzyme-like protein (EgtB), a glutamine amidotransferase (EgtC), a histidine methyltransferase (EgtD), and an unrelated β-lyase from Erwinia tasmaniensis to replace the pyridoxal 5-phosphate binding protein (EgtE), because the recombinant production of soluble EgtE protein failed (see, J. Am. Chem. Soc. 2010, 132:6632-6633).
Thus far, only 3 genes coding for EgtB, EgtC, and EgtD have been identified for the production of ergothioneine in vitro. A putative gene for EgtE remains uncharacterized either in vitro or in vivo. To date, no microbial production using the above genes to engineer the mycobacterial ergothioneine metabolic pathway in E. coli has been reported despite various attempts at bio-conversion. Also, although various fungal and mycobacterial sources are available for the ergothioneine extraction, the yields are too low to be commercially viable for industrial production of ergothioneine. Accordingly, there exists a need for producing ergothioneine.